Double-structured tissue implant and a method for preparation and use thereof

ABSTRACT

A double-structured tissue implant and a method for preparation and use thereof for implantation into tissue defects. The double-structured tissue implant comprising a primary scaffold and a secondary scaffold consisting of a soluble collagen solution in combination with a non-ionic surfactant generated and positioned within the primary scaffold. A stand alone secondary scaffold implant or unit. A process for preparation of the double-structured implant or the stand alone secondary scaffold comprising lyophilization and dehydrothermal treatment.

This application claims priority of the Provisional application Ser. No.60/958,401, filed Jul. 3, 2007 and incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of Invention

The current invention concerns a double-structured tissue implant and amethod for preparation and use thereof for implantation into tissuedefects. In particular, the invention concerns a double-structuredtissue implant comprising a primary scaffold and a secondary scaffoldgenerated and positioned within the primary scaffold. The primaryscaffold is a porous collagen-comprising material having randomly ornon-randomly oriented pores of substantially homogeneous defineddiameter. Under the most favorable conditions, the pores are verticallyoriented and represent a high percentage of the porosity of thescaffold. The secondary scaffold is generated within the primaryscaffold by introducing a composition comprising a soluble collagensolution in combination with a non-ionic surfactant into the pores ofthe primary scaffold and solidifying said composition within said poresusing a novel process of the invention.

The process for preparation of the double-structured tissue implantcomprises introducing a solution of components used for generation ofthe secondary scaffold into the primary scaffold and processing using atleast precipitation, lyophilization and dehydrothermal treatment steps.

Each the primary and secondary scaffold independently, or together, mayprovide a structural support for cells and/or each separately maycontain cells, drugs or growth modulators.

When formed, the double-structured tissue implant has improvedproperties, such as stability, resistance to shrinkage, swelling ordissolution, improved wetting, storageability and longer shelf-life ascompared to the properties of each individual scaffold or the composite.

Furthermore, the double-structured tissue implant provides an increasedsurface area for cell adhesion, growth and differentiation withoutcompromising the porosity of the implant.

BACKGROUND AND RELATED DISCLOSURES

Collagen matrices for use as an implant for repair of cartilage defectsand injuries are known in the art. Of a particular interest is ahoneycomb structure developed by Koken Company, Ltd., Tokyo, Japan,under the trade name Honeycomb Sponge, described in the Japanese patentJP3170693, hereby incorporated by reference. Other patents related tothe current subject disclose collagen-based substrates for tissueengineering (U.S. Pat. No. 6,790,454) collagen/polysaccharide bi-layermatrix (U.S. Pat. No. 6,773,723), collagen/polysaccharide bi-layermatrix (U.S. Pat. No. 6,896,904), matrix for tissue engineering formedof hyaluronic acid and hydrolyzed collagen (U.S. Pat. No. 6,737,072),method for making a porous matrix particle (U.S. Pat. No. 5,629,191)method for making porous biodegradable polymers (U.S. Pat. No.6,673,286), process for growing tissue in a macroporous polymer scaffold(U.S. Pat. No. 6,875,442), method for preserving porosity in porousmaterials (U.S. Pat. No. 4,522,753), method for preparation ofcollagen-glycosaminoglycan composite materials (U.S. Pat. No.4,448,718), procedures for preparing composite materials from collagenand glycosaminoglycan (U.S. Pat. No. 4,350,629) and a crosslinkedcollagen-mucopolysaccharide composite materials (U.S. Pat. No.4,280,954).

However, many of the above disclosed structures have uncontrolledparameters such as uneven and uncontrolled porosity, uneven density ofpores, uneven sizes of the pores and random distribution of pores withinthe support matrix. Such uncontrolled parameters lead to usable porestructures that represent only a small percentage of the total implant.Additionally, when introduced into tissue defects or cartilage lesionsduring the surgery, these structures are difficult to handle as they areunstable and do not have appropriate wetting properties in that they canshrink or swell and are not easily manipulated by the surgeon.

For a tissue implant to be suitable for implantation, particularly forimplantation into the cartilage lesion, the implant needs to be stable,easily manipulated, easily stored in sterile form and have a longshelf-life.

In order to provide a more uniform and sterically stable supportstructure for implantation into a tissue defect or cartilage lesion,inventors previously developed a collagen matrix having narrowly definedsize and density of pores wherein the pores are uniformly distributed,vertically oriented and non-randomly organized. This matrix is disclosedin the co-pending patent application Ser. No. 11/523,833, filed on Sep.19, 2006, hereby incorporated by reference in its entirety.Additionally, the acellular matrix suitable to be used as the primaryscaffold is described in the priority application Ser. No. 10/882,581,filed on Jun. 30, 2004, issued as U.S. Pat. No. 7,217,294, on May 15,2007, hereby incorporated in its entirety.

However, even with the above-described improvements, a solution toproblems faced by the surgeon during surgery is still lacking. Apracticality needed for routine use of the tissue implants, such as, forexample, the articular cartilage implants by the orthopedic surgeons,where the implant needs to be readily available, manipulatable,wettable, stable, sterile and able to be rapidly prepared and used forimplantation, is still not achieved. All the previously described andprepared matrices or scaffolds require multiple steps before they arefully implantable.

Thus, it would be advantageous to have available an implant that wouldbe easily manufactured and packaged, would be stable for extendedshelf-life, would be easily manipulatable and rapidly wettable uponintroduction into the lesion, could provide a support for cell migrationor seeding and that could have, additionally, pre-incorporated drug ormodulator in at least one portion of the implant. The implant shouldalso allow the surgeon to introduce a drug or modulator during thesurgical procedure.

It would also be an advantage to provide a secondary scaffold with anincreased area of internal membranes which while not interfering withcell migration and nutrient exchange, nevertheless, would provide asubstrate favorable to cell adhesion, growth and migration.

It is, therefore, a primary object of this invention to provide adouble-structured tissue implant comprising of a primary scaffold and asecondary scaffold where each scaffold of the implant can assumedifferent function, be incorporated with cells, different drugs ormodulators and/or be selectively chosen for performing differentfunction following the implantation.

The current invention provides such double-structured scaffold and/or amethod for use and fabrication thereof by providing a first scaffoldcomprising a sterically stable and biocompatible support structure,preferably made of Type I collagen, having defined pore sizes anddensity with said pores organized vertically and a second scaffoldwherein said second scaffold is formed within said pores of said firstscaffold. The double-structured scaffold of the invention is stable,resistant to shrinkage, swelling and dissolution, rapidly wettable,prepared in the sterile storageable form having a long-shelf life thatcan be easily surgically delivered and easily manipulated.

All patents, patent applications and publications cited herein arehereby incorporated by reference.

SUMMARY

One aspect of the current invention is a double-structured tissueimplant, process for preparation thereof and a method for use thereof.

Another aspect of the current invention is a collagen-baseddouble-structured tissue implant comprising a primary scaffold and asecondary scaffold wherein said secondary scaffold is a qualitativelydifferent structure formed within a confines of the primary scaffold.

Another aspect of the current invention is a collagen-based primaryporous scaffold having vertically oriented open pores of substantiallyhomogeneous pore size, said primary scaffold suitable for incorporationof a secondary scaffold wherein said secondary scaffold is incorporatedinto said primary scaffold by introducing a basic solution comprisingcollagen and a non-ionic surfactant into said primary scaffold andsubjecting said primary scaffold incorporated with said basic solutionfor the secondary scaffold to a process comprising precipitation,lyophilization and dehydrothermal treatment.

Still yet another aspect of the current invention is a double-structuredtissue implant having two distinct qualitatively different structureswherein each of the structures may be independently seeded with cells orwherein one or both structures of the implant may comprise apharmaceutical agent or growth modulator.

Still another aspect of the current invention is a secondary scaffold asa stand alone implant or unit, said secondary scaffold prepared from theneutralized basic solution comprising collagen and a surfactantsubjected to lyophilization and dehydrothermal treatment.

Yet another aspect of the current invention is a method for use of adouble-structured tissue implant or the stand alone secondary scaffoldimplant for implantation into a tissue defect or cartilage lesionwherein said implant, in dry or wet form, is implanted into said defector lesion during surgery.

Another aspect of the current invention is a process for preparation ofa double-structured implant by providing a primary porous scaffoldprepared from a biocompatible collagen material wherein said scaffoldhas a substantially homogeneous defined porosity and uniformlydistributed randomly and non-randomly organized pores of substantiallythe same size of defined diameter of about 300±100 μm, wherein saidprimary scaffold is brought in contact with a soluble collagen basedsolution comprising at least one non-ionic surfactant (basic solution),wherein such solution is introduced into said pores of said primaryscaffold, stabilized therein by precipitation or gelling, dehydrated,lyophilized and dehydrothermally processed to form a distinctlystructurally and functionally different second scaffold within saidpores of said primary scaffold.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A is a photograph of the lyophilized double-structured tissueimplant (DSTI) that shows the porous nature and the sturdiness of theimplant prior to rehydration. FIG. 1B is a photograph of thedouble-structured tissue implant (DSTI) packaged in a sterile form readyfor delivery.

FIG. 2 is a photomicrograph of a primary scaffold showing pores havingsubstantially the same size (4× magnification), said scaffold used asthe foundation and structural support for preparation of thedouble-structured tissue implant. As shown, the primary scaffold has ahoneycomb structure of relative uniform pore size and equaldistribution.

FIG. 3 is a photomicrograph of a primary scaffold loaded with a solublecollagen/PLURONIC® surfactant composition (basic solution) beforeprecipitation and processing with dehydrothermal treatment (4×magnification) wherein the basic solution is prepared and applied as anaqueous gel which evenly fills the pores.

FIG. 4A is a photomicrograph of a rehydrated double-structured tissueimplant (DSTI) showing a primary and secondary scaffold (4×magnification). FIG. 4A demonstrates the formation of thedouble-structured implant, where the secondary scaffold is observed fromthe fibrous-like diffraction pattern present within the pores of theprimary scaffold. The diffraction pattern is created from thepolymerization of the collagen within the pores. The collagen fibersinterdigitate within the pores and among the pores.

FIG. 4B is a photomicrograph of the dehydrated double-structured tissueimplant prior to implantation showing a primary and secondary scaffolds(4× magnification). Similarly to FIG. 4A, FIG. 4B shows thedouble-structured implant wherein the secondary scaffold is seen as thefibrous-like diffraction pattern present within the pores of the primaryscaffold.

FIG. 5 is a photomicrograph of a double-structured tissue implant seededwith chondrocytes after 14 days in culture showing a primary scaffold, asecondary scaffold and chondrocytes attached to or embedded within thesecondary scaffold localized in the pores of the primary scaffold (10×magnification). The DSTI shown in FIG. 5 was dehydrothermally treatedand subsequently has undergone rehydration with a phosphate bufferedsaline and seeding with chondrocytes that were maintained in cultureover a period of 14 days. The cultured chondrocytes are shown to adhereto the fibrous secondary scaffold, as well as aggregate within thepores.

FIG. 6 is a graph illustrating collagen retention in phosphate bufferedsaline, and thus resistance to dissolution from three separatedouble-structured tissue implants (DSTIs) compared to a compositeconsisting of a primary scaffold loaded with a composition for asecondary, scaffold but not lyophilized or dehydrothermally processed(Composite). FIG. 6 demonstrates the structural stability of thecollagen network present in the double-structured implant (DSTI) as afunction of time in an aqueous buffered saline solution. The datademonstrate that during the first day following the rehydration, thereis very little dissolution of collagen from the DSTI and that theretention of collagen is almost 100% during the initial first criticalhour. On the other hand, the dissolution from the Composite in theinitial hour is much higher and retention drops immediately toapproximately 96% during that same critical first hour. FIG. 6 thusclearly demonstrates a stability of the DSTI.

FIG. 7 is a graph illustrating percent of surface area change ofdouble-structured tissue implant (DSTI) and a composite (Composite) of aprimary scaffold loaded with a composition for a secondary scaffoldbefore lyophilization and dehydrothermal processing, from 1 to 24 hours.The implants were rehydrated with an aqueous phosphate buffered salineand maintained in culture for eight days. Results show that there is aninsignificant small change in the surface area during the first hourfollowing the rehydration in both DSTI and Composite. This figureconfirms that in the double-structured tissue implants (DSTI) subjectedto the dehydrothermal treatment there is a very small change in thesurface area and therefore no shrinkage or swelling following therehydration.

FIG. 8 is a graph showing production of S-GAG/DNA by chondrocytes seededin double-structured tissue implant (DSTI) and in a composite(Composite) comprising a primary scaffold loaded with a composition fora secondary scaffold before lyophilization and dehydrothermalprocessing, after 14 days in culture. FIG. 8 demonstrates that secondaryscaffold supports the growth of cells and deposition of extracellularmatrix measured here as sulfated glycosaminoglycan. A comparison betweenthe double-structured tissue implant and the Composite showed comparableresults with little evidence of significant steric hindrance due to theadded structural components.

FIG. 9 is a schematic illustration of two treatment protocols forimplantation of the DSTI into cartilage lesions using double-structuredtissue implants. FIG. 9A shows the treatment with DSTI withoutmicrofracture, FIG. 9B shows treatment with microfracture pretreatment.FIGS. 9A and 9B demonstrate how the double secondary scaffold will beapplied in a surgical operating room setting. The initially oversizeddouble-structured tissue implant (DSTI) is first cut and trimmed tomatch the defect. A physiologically acceptable tissue adhesive isapplied to the defect to coat the defect site (FIG. 9A) or thesubchondral plate of the defect is penetrated by microfracture, adhesiveis applied between microfracture penetrations (FIG. 9B) and the precutsized DSTI is placed into the defect. The DSTI is then rehydrated with aphysiologically acceptable solution optionally containing agents thatstimulate healing. The DSTI is then sealed within the defect usinganother layer of the tissue adhesive to form a final bonding.

DEFINITIONS

As used herein:

“Primary scaffold” means a porous honeycomb, sponge, lattice or anotherstructure made of collagen or collagen based material having randomly ornon-randomly oriented pores of substantially homogeneous defineddiameter. Under the most favorable conditions, the pores are verticallyoriented and represent a high percentage of the porosity of thescaffold.

“Secondary scaffold” means a collagen based structured prepared from acollagen or collagen based compound in the presence of a non-ionicsurfactant. The secondary scaffold is generated within the primaryscaffold by introducing a composition comprising a soluble collagensolution in combination with a non-ionic surfactant (basic solution)into the pores of the primary scaffold and solidifying said compositionwithin said pores using a novel process of the invention.

“Basic solution” means a solution comprising a collagen in admixturewith a surfactant, preferably PLURONIC®-type surfactant, neutralized tothe pH of about 7.4. Basic solution is used for preparation of thesecondary scaffold.

“Composite” means a primary scaffold loaded with a compositioncomprising a precipitated or gelled soluble collagen in combination witha non-ionic surfactant (basic solution). The composite is in a hydratedform because the basic solution is added in a fluid form as a gel,suspension or solution.

“Lyophilized composite” means the hydrated “composite”, as definedabove, that is subsequently subjected to a dehydration andlyophilization step.

“Double-structured tissue implant” or “DSTI” means a tissue implantprepared according to a process of the invention wherein the primaryscaffold is loaded with a basic solution thereby forming a compositethat is subsequently subjected to precipitation, dehydration andlyophilization to obtain lyophilized composite that is subsequentlytreated with dehydrothermal (DHT) treatment to result in a stabledouble-structured tissue implant.

“Surfactant” means a non-ionic or ionic surfactant polymer. Suitablesurfactants, such as PLURONIC®-type polymers or TRITON®-type polymers,are non-ionic co-polymer surfactants consisting of polyethylene andpolypropylene oxide blocks. TRITON®-type surfactants are commerciallyavailable derivatized polyethylene oxides, such as for example,polyethylene oxide p-(1,1,3,3-tetramethylbutyl)-phenyl ether, knownunder its trade name as TRITON®-X100. Other TRITON®-type surfactantsthat may be suitable for use in the instant invention are TRITON® X-15,TRITON® X-35, TRITON® X-45, TRITON® X-114 and TRITON® X-102. TRITON®surfactant are commercially available from, for example, Union Carbide,Inc. PLURONIC®-type surfactants are commercially available blockco-polymers of polyoxyethylene (PEO) and polyoxypropylene (PPO) havingthe following generic organization of polymeric blocks: PEO-PPO-PEO(Pluronic) or PPO-PEO-PPO (Pluronic R). Exemplary PLURONIC®-typesurfactants are PLURONIC® F68, PLURONIC® F127, PLURONIC® F108, PLURONIC®F98, PLURONIC® F88, PLURONIC® F87, PLURONIC® F77, PLURONIC® F68,PLURONIC® 17R8 and PLURONIC® 10R8.

“The porosity” means a pore size defined by the diameter of holes withinthe primary scaffold as well as density of the pore distribution as afunction of cross-sectional area in millimeters. Porosity is defined asa total volume of pores relative to the implant.

“Substantially homogeneous” means at least 85-99% homogeneity.Preferable homogeneity is between 95% and 99%.

“Substantially homogeneous porosity” means that a pore size and diameteris within pore size range of about 300±100 μm, preferably 300±50 μm, indiameter.

“Wettability” means an ability to quickly absorb a fluid into the DSTIwithout changes in the size and shape of the implant.

“Shrinkage” means a volumetric reduction in surface area in alldimensions of a double-structured tissue implant.

“Swelling” means a volumetric increase of a surface area in alldimensions of a double-structured tissue implant.

“Dissolution” means the act of a solid matter being solubilized by asolvent.

“Rehydration” means the act of hydrating, wetting or rewetting adehydrated composite, lyophilized composite, stand alone secondaryscaffold or double-structured tissue implant.

“Dehydrothermal treatment” means removing water at low pressure and athigh temperature for cross-linking of polymers.

“Top surface” means an apical or synovial side of the matrix turnedtoward the joint.

“Bottom surface” means basal, closest to bone surface of the matrix.

“Chondrocytes” means the cells naturally residing in articularcartilage.

“S-GAG” means sulfated glycosaminoglycan.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is directed to a double-structured tissue implantsuitable for implantation into a tissue lesion, to a process forpreparation of said implant and to the method for use of said implant ina clinical setting for repair of tissue lesions. The double-structuredimplant has improved properties compared to a single structured implantand provides for variability in use.

The double-structured tissue implant (DSTI) comprises a primarycollagen-based structure, hereinafter called a primary scaffold, and asecondary collagen-based structure, hereinafter called a secondaryscaffold. The two scaffolds are structurally and functionally different.Each scaffold is prepared from a different collagen-based compositionwith the primary scaffold prepared first and the secondary scaffoldprepared by introducing a different collagen-based composition withinthe primary scaffold thereby forming a composite structure comprising aprimary scaffold incorporated with a composition (basic solution) forpreparation of said secondary scaffold. The composite structure is thensubjected to a lyophilization and dehydrothermal treatment processaccording to the invention.

The resulting double-structured implant thus contains a structurallydifferent secondary scaffold incorporated into and localized withinpores of the primary scaffold.

The actual photograph of a double-structured tissue implant is seen inFIG. 1 in two manifestations.

FIG. 1A shows a close-up view of a DSTI, as delivered to a surgeon inready form for implantation. Before implanting, the surgeon cuts thepiece of the implant to size and shape corresponding to the size andshape of the defect, places the precut section into the defect andrehydrates the implant with sterile phosphate buffered saline or anotherphysiologically acceptable solution. The precut implant is suitable forplacement within a full thickness defect of a tissue, and particularlyarticular cartilage. Once in place, the double-structured scaffold canbe held in place by suture or biologically acceptable adhesive.

FIG. 1B shows the double-structured tissue implant as a dry sterileproduct enclosed in a shipping container within sterile packaging. Thepackaged product is available as an off-the-shelf DSTI for implantationin clinical settings.

Double-Structured Tissue Implant

A double-structured tissue implant (DSTI) comprises two separatelyprepared components, namely the primary scaffold that provides astructural support for the secondary scaffold incorporated within theprimary scaffold.

The Primary Scaffold

The primary scaffold is a collagen-based matrix prepared as a honeycomb,lattice, sponge or any other similar structure made of a biocompatibleand/or biodegradable collagen containing material of defined density andporosity that is pliable, storageable and, most importantly, highlyporous.

Typically, the primary scaffold is prepared from collagen,collagen-containing composition or collagen containing a polymer.Representative compounds suitable for preparation of the primaryscaffold are a Type I collagen, Type II collagen, Type IV collagen,gelatin, collagen containing agarose, collagen containing hyaluronan,collagen containing proteoglycan, glycosaminoglycan, glycoprotein,glucosamine or galactosamine, collagen containing fibronectin, collagencontaining laminin, collagen containing a bioactive peptide growthfactor, collagen containing cytokine, collagen containing elastin,collagen containing fibrin, collagen containing polylactic, polyglycolicor polyamino acid, collagen containing polycaprolactone, collagencontaining polypeptide, or a copolymer thereof, each alone or incombination.

Additionally, the primary scaffold may be prepared from the collagenprecursors, such as, for example, peptide monomers, such as alpha 1(type I), and alpha 2 (type I) collagen peptide or alpha 1 (type I)alpha 2 (type I) peptides, alone or in combination, or from acombination of precursors, such as 2 (alpha 1, type I) peptide and 1(alpha 2, type I) peptide.

The collagen containing material used for preparation of the primaryscaffold may further be supplemented with other compounds, such aspharmaceutically acceptable excipients, surfactants, buffers, additivesand other biocompatible components.

Preferably, the primary scaffold of the invention is prepared fromcollagen and most preferably from Type I collagen or from a compositioncontaining Type I collagen.

In one embodiment, the primary scaffold is a structure containing aplurality of narrowly defined randomly or non-randomly organized poreshaving a substantially homogeneous narrowly defined size and diameterTHAT are uniformly distributed through the scaffold, dividing thescaffold space into columns or pore network. The exemplary primaryscaffold is described in the co-pending application Ser. No. 11/523,833,filed on Sep. 19, 2006, herein incorporated by reference in itsentirety.

In another embodiment, the primary scaffold may be the Type Icollagen-based support matrix that is a collagen-based porous honeycomb,sponge, lattice, sponge-like structure or honeycomb-like lattice ofdefined porosity having randomly or non-randomly organized pores ofvariable pore diameters such as described in, for example, applicationsSer. No. 10/882,581, filed on Jun. 30, 2004, issued as U.S. Pat. No.7,217,294 on May 15, 2007, herein incorporated by reference.

In yet another embodiment the primary scaffold is a honeycomb collagenmatrix developed by Koken Company, Ltd., Tokyo, Japan, under the tradename Honeycomb Sponge, described in the Japanese patent JP3170693,hereby incorporated by reference.

The primary scaffold according to the invention has, preferably, asubstantially defined pore size in diameter and pore density in randomlyor non-randomly organized manner that creates an apical (top) or basal(bottom) surface to the implant where the sizes and diameters of thepores on both the apical or basal surface are substantially the same.When used as a primary scaffold only, the scaffold provides conditionsfor a sterically-enhanced enablement of cells. Chondrocytes, forexample, produce an extracellular matrix comprising glycosaminoglycanand Type II collagen within said implant in ratios characteristic for anormal healthy articular cartilage.

A microphotograph of the primary scaffold is shown in FIG. 2. FIG. 2 isa representation of one embodiment of the primary scaffold that isobtained and used as the structural foundation for preparation of thedouble-structured tissue implant. As seen in FIG. 2, the primaryscaffold has a porous honeycomb structure of relatively uniform poresize and equal distribution.

A secondary scaffold structure is generated within the pores of theprimary scaffold. To that end, the primary scaffold is loaded with acomposition suitable for preparation of the secondary scaffold (basicsolution). Such composition comprises a soluble collagen,collagen-containing or collagen-like mixture, typically of Type Icollagen, in combination with a non-ionic surfactant. The primaryscaffold loaded with the composition for formation of the secondaryscaffold is shown in FIG. 3.

FIG. 3 is a microphotographic representation of the primary scaffoldthat has been loaded with the solubilized collagen/surfactantcomposition that forms a basis for formation of the secondary scaffold.Such composition is prepared as a solution, suspension or as an aqueousgel at a dilute acidic pH and is further neutralized to pH 7.4 (basicsolution). The basic solution is then applied to or is loaded into theprimary scaffold such that it evenly fills the porous structure of theprimary scaffold.

B. The Secondary Scaffold

The secondary scaffold is created or generated within the pores of theprimary scaffold. The secondary scaffold is a qualitatively differentstructure formed within the confines of the first scaffold.

The secondary scaffold is generated by a process comprising preparing asoluble collagen-based composition as described below, furthercomprising a suitable non-ionic or ionic surfactant (basic solution).

The secondary scaffold comprises a collagen, methylated collagen,gelatin or methylated gelatin, collagen-containing and collagen-likemixtures, said collagen being typically of Type I or Type II, eachalone, in admixture, or in combination and further in combination with asurfactant, preferably a non-ionic surfactant. The suitable surfactantis preferably a polymeric compound such as a PLURONIC®-type polymer.

Additionally, the secondary scaffold may be used independently of theprimary scaffold as said secondary scaffold stand alone implant or unitwhere the basic solution can be introduced into a mold or container andsubjected to precipitation, lyophilization and dehydrothermal treatment.

In preparation of the DSTI, said composition suitable for generation ofthe secondary scaffold within the primary scaffold is then brought intocontact with a primary scaffold structure by absorbing, wicking, soakingor by using a pressure, vacuum, pumping or electrophoresis, etc., tointroduce said composition for the secondary scaffold into the pores ofthe primary scaffold. In alternative, the primary scaffold may beimmersed into the composition for the secondary scaffold.

C. Double-Structured Tissue Implant

The double structured tissue implant (DSTI) is prepared by treating theprimary scaffold loaded with a combination of the soluble collagen andnon-ionic surfactant and processes according to the process forpreparation of the DSTI described below in Scheme 1.

Briefly, the primary scaffold is loaded with the collagen/surfactantcombination, precipitated or gelled, washed, dried, lyophilized anddehydrothermally treated to solidify and stabilize the secondaryscaffold within the pores of the primary scaffold.

A rehydrated double-structured tissue implant is seen in FIG. 4, wherethe primary scaffold pores are seen as delineating black lines. Thepores are filled with the secondary scaffold.

FIG. 4A and FIG. 4B show the rehydrated and dry forms of thedouble-structured tissue implant, where the secondary scaffold isobserved from the fibrous-like diffraction pattern present within thepores of the primary scaffold. The diffraction pattern occurs due to thepolymerization of the collagen within the pores. The collagen fibersinterdigitate within the pores and among the pores.

The double-structured tissue implant can be seeded with cells, loadedwith pharmaceutical agents, drugs or growth modulators. Additionally andpreferably, the two of its distinct components, namely the primaryscaffold and the secondary scaffold, can each be independently loadedwith living cells, cell suspension, with a pharmaceutically effectiveagent or agents or with growth modulator. These may be loaded into theimplant individually or in any possible combination, such as, forexample, where the cells may be introduced into one component and thedrug into the second component, or the drug into one component and themodulator into the second component and/or any variation thereof.

The DSTI loaded with chondrocytes is shown in FIG. 5 wherein the cellsare attached to the secondary scaffold.

FIG. 5 is a microphotograph of the dehydrothermally treateddouble-structured tissue implant that has undergone rehydration and hasbeen treated with chondrocyte deposition and maintained in culture overa period of several days. FIG. 5 shows cells adherent to the fibroussecondary scaffold, as well as being present as aggregates within thepores.

D. The Drug Containing Double-Structured Tissue Implant

The double-structured implant of the invention provides for avariability of uses. One embodiment of the use is the double-structuredtissue implant containing the pharmaceutical agent, drug, growthmodulator, growth hormone, mediator, enzyme promoting cellincorporation, cell proliferation or cell division, pharmaceuticallyacceptable excipient, additive, buffer etc.

The drug may be introduced separately into the primary scaffold, intothe secondary scaffold or be added at a time of rehydration to acomposition for the secondary scaffold before its processing.

The pharmaceutical agents, drugs or modulators are selected from thegroup consisting of:

growth and morphogenic factors, such as, for example, transforminggrowth factor, insulin-like growth factor 1, platelet-derived growthfactor, bone morphogenetic proteins (bmps);

cytokines, such as, for example, interleukins, chemokines, macrophagechemoattractant factors, cytokine-induced neutrophil chemoattractants(gro-1), integral membrane proteins such as integrins and growth factorreceptors;

membrane associated factors that promote growth and morphogenesis, suchas, for example, repulsive guidance molecules;

cell attachment or adhesion proteins, such as, for example, fibronectinand chondronectin;

hormones, such as, for example, growth hormone, insulin and thyroxine;

pericellular matrix molecules, such as perlecan, syndecan, smallleucine-rich proteoglycans and fibromodulin;

nutrients, such as, for example, glucose and glucosamine;

nucleic acids, such as, for example, RNA and DNA;

anti-neoplastic agents, such as, for example, methotrexate andaminopterin;

vitamins, such as, for example, ascorbate and retinoic acid;

anti-inflammatory agents, such as, for example, naproxen sodium,salicylic acid, diclofenac and ibuprofen;

enzymes, such as, for example, phosphorylase, sulfatase and kinase; and

metabolic inhibitors, such as, for example, RNAi, cycloheximide andsteroids.

These, and other similar compounds and/or compounds belonging to theabove-identified groups may be added individually or in combination to aprimary scaffold, to a secondary scaffold, to a composition (basicsolution) for formation of the secondary scaffold or to the lyophilizedcomposite or DSTI before, during or after implantation.

Addition of agents such as growth factors, cytokines and chemokines willincrease cell migration, cell growth, will maintain or promoteappropriate cell phenotype and will stimulate extracellular matrixsynthesis. Loading the scaffold with anti-inflammatory agents or otherdrugs can provide a local site-specific delivery system.

The range of concentration of the added drug or compound depends on thedrug or compound and its function and it extends from picograms tomilligrams.

E. Secondary Scaffold as a Stand Alone Implant

In one embodiment, the secondary scaffold may be generated as a standalone structure. In this regard, a composition comprising a solublecollagen, methylated collagen, gelatin or methylated gelatin in anacidic solution further comprising a non-ionic surfactant is subjectedto neutralization, precipitation, dehydration, lyophilization anddehydrothermal treatment under conditions as described in the Scheme 1.

As described for the double structured tissue implant, the secondaryscaffold as a stand alone implant may be similarly seeded with cells andmay optionally contain a pharmaceutical agent, growth modulator oranother compound, as described above.

The process for preparation of the stand alone secondary scaffold ismodified to the extent that the composition for preparation of thesecondary scaffold (basic solution) is placed into a container or formsuitable to permit gelling, precipitation, dehydration, lyophilizationand dehydrothermal treatment.

The stand alone secondary scaffold is used for implantation in the samemanner as described for double-structured tissue implant. The standalone secondary scaffold is useful in the healing of tears in cartilageor skeletal tissues, such as, for example, the meniscus where it can becharged and/or supplemented with all of the tissue factors and cells,such as meniscal fibroblasts.

The stand alone secondary scaffold can be used in a similar fashion forbone, tendon and ligament repair.

Components and conditions suitable for preparation of the secondaryscaffold stand alone structure and evaluation of its performance usingcell viability are seen in Table 1.

TABLE 1 Pluronic Collagen F127 Dissolution Cell conc. conc. DHT, 6 hRehydration stability in viability (mg/ml) (mg/ml) at 140° C. time (s)PBS at 37° C. (%) Precipitated or gelled using ammonia vapor 3 0.25 Y 12Stable — 3 1 Y 13 Stable — 3 1 Y <10 Stable 97 3 1 N <10 Dissolved — 3 3Y <10 Stable 98 3 3 N <10 Dissolved — 2.9 0.29 Y 6 Stable — 2.9 0.29 N 3Dissolved — 2 0.165 Y 7 Stable — 2 0.33 Y 3 Stable — 2 0.67 Y 2 Stable —2 0.25 Y 15 Stable — 2 0.5 Y 7 Stable — 2 0.1 Y 4 Stable — 2 2 Y <10Stable 99 2 2 N <10 Dissolved — 1.5 0.15 Y <10 Dissolved — 1.5 0.15 N 16Dissolved — Precipitated or gelled using NaOH 2.3 0.05 Y 23 Stable — 2.30.1 Y 19 Stable — 2.3 0.23 Y 15 Stable — 2.4 0.24 Y 5 Stable —

Table 1 summarizes experimental conditions used for determination ofoptimization of conditions for preparation of a secondary scaffold. Theconditions tested and evaluated were a collagen concentration,surfactant concentration, temperature and time for dehydrothermaltreatment (DHT), rehydration time, stability of the DSTI determined bydissolution of the secondary scaffold in phosphate buffer saline at 37°C., and cell viability.

The secondary scaffold was precipitated in the presence of ammonia vaporor ammonia aqueous solution or in the presence of 0.1M NaOH.

Results seen in Table 1 show the effectiveness of dehydrothermaltreatment for preparation of secondary scaffolds, in terms of achievingthe stability of the secondary scaffold, its fast rehydration andassuring cell viability within the secondary scaffold.

As seen from the results summarized in Table 1, following ammoniaprecipitation of the collagen, dissolution stability was not observed inthe absence of dehydrothermal treatment in spite of varying collagen andsurfactant concentrations. In instances where stability was achieved,there was excellent cell loading and viability at collagenconcentrations greater than 1-5 mg/ml and at surfactant concentrationsof 1, 2 and 3 mg/ml.

In an alternate approach, the precipitation of collagen byneutralization with NaOH, in the presence of Pluronic and subject toDHT, detected a rapidly rehydrating and stable secondary scaffold.

These results clearly show that the properties of the secondary scaffoldalone or the secondary scaffold incorporated into the DSTI may beconveniently optimized to achieve fast rehydration time, dissolutionstability and excellent cell loading and cell viability up to 99% withinthe secondary scaffold.

F. Surfactants

Improved properties of the DSTI, such as its rapid wettability andresistance to shrinkage, swelling and dissolution, are due to a presenceof a secondary scaffold as a distinct functional entity.

The secondary scaffold prepared according to the process of theinvention requires, as an essential part, a presence of a surfactant,preferably a non-ionic or, in some instances, even an ionic surfactant.The surfactant, preferably the non-ionic surfactant of type such asTRITON® or PLURONIC®, preferably PLURONIC® F127, is an essentialcomponent of a composition used for preparation of the secondaryscaffold, or micellar substrate bound to the implant. The presence ofthe surfactant improves stability and particularly wettability andrehydration properties of the implant without causing its shrinkage orswelling.

Suitable surfactants, such as PLURONIC®-type polymers or TRITON®-typepolymers, are non-ionic co-polymer surfactants consisting ofpolyethylene and polypropylene oxide blocks.

TRITON®-type surfactants are commercially available derivatizedpolyethylene oxides, such as for example, polyethylene oxidep-(1,1,3,3-tetramethylbutyl)-phenyl ether, known under its trade name asTRITON®-X100. Other TRITON®-type surfactants that may be suitable foruse in the instant invention are TRITON® X-15, TRITON® X-35, TRITON®X-45, TRITON® X-114 and TRITON® X-102. TRITON® surfactant arecommercially available from, for example, Union Carbide, Inc.

PLURONIC®-type surfactants are commercially available block co-polymersof polyoxyethylene (PEO) and polyoxypropylene (PPO) having the followinggeneric organization of polymeric blocks: PEO-PPO-PEO (Pluronic) orPPO-PEO-PPO (Pluronic R). Exemplary PLURONIC®-type surfactants arePLURONIC® F68, PLURONIC® F127, PLURONIC® F108, PLURONIC® F98, PLURONIC®F88, PLURONIC® F87, PLURONIC® F77, PLURONIC® F68, PLURONIC® 17R8 andPLURONIC® 10R8.

The most preferred non-ionic surfactant of PLURONIC®-type suitable foruse in the invention is a block co-polymer of polyoxyethylene (PEO) andpolyoxypropylene (PPO) with two 96-unit hydrophilic PEO blockssurrounding one 69-unit hydrophobic PPO block, known under its tradename as PLURONIC® F127. PLURONIC® surfactants are commercially availablefrom BASF Corp.

E. Properties of the Double-Structured Tissue Implant

The DSTI of the invention has distinctly improved properties whencompared to the primary scaffold alone, to the secondary scaffold aloneor to a composite loaded with a composition for preparation of thesecondary scaffold (Composite), unprocessed, or to the Composite thathas been dehydrated and lyophilized (Lyophilized Composite).

Typically, a tissue implant is implanted into a tissue defect during asurgery. Also typically, such surgery has a time-limit on implantationthat has preferably about one hour window when the implant is placedinto the defect. For these reasons, it is important that a specificationfor an implantable double-structured tissue implant provides stability,resistance to change in shape, size and shrinkage or swelling,resistance to dissolution, consistency with respect to pore sizepermitting an ingrowth of cells into the implant and conditions forformation of extracellular matrix within the implant. The DSTI appearsto have all the above properties.

Furthermore, a presence of the secondary scaffold improves the functionof the DSTI by providing a multitude of small membranous substrateswhich can provide cell anchorage and phenotype stability whilepreserving the through porosity of the overall implant, thereby allowingnutrients and growth factors and migratory cells to permeate theimplant.

A. Stability of the Double-Structured Tissue Implant

From the point of view of the implantability, stability of the implantis one of the major requirements. The implant stability depends onseveral factors. There must be low or, preferably, no initialdissolution of collagen from the implant into the physiological fluidsand there must be low or preferably no change in size and shape of theimplant following rehydration or wetting before, during or after surgeryprior to biodegradation in situ.

1. Collagen Retention and Resistance to Dissolution

One of the most important requirements for the implant is its resistanceto dissolution of its components upon wetting and rehydration of saidimplant during implantation during preparation of the implant forimplantation and subsequently also after implantation. A low dissolutionor, preferably, no dissolution of the collagen component from theimplant into the physiologic solution after or before placement of theimplant into the tissue defect, and into an interstitial fluid, plasmaor blood following the surgery, under normal physiological conditionsensures continued functionality of the implant following itsimplantation into the tissue defect, such as, for example into thecartilage lesion. Low or no dissolution of collagen from the implantmeans the high retention of the collagen within the implant.

FIG. 6 demonstrates the structural stability of the collagen networkpresent in the double-structured tissue implant as a function of timefollowing rehydration of the implant with an aqueous phosphate bufferedsaline solution by comparing collagen retention, in percent, withinDSTI, to collagen retention within an unprocessed composite (Composite)comprising a primary scaffold loaded with a composition for a secondaryscaffold (basic solution) but not lyophilized or dehydrothermallyprocessed.

Results seen in FIG. 6 demonstrate the structural stability of thecollagen network present in the double-structured tissue implant as afunction of time in an aqueous buffered saline solution. The datademonstrate that during the first day following the rehydration, thereis very little dissolution of collagen from the DSTIs (D#1-3) and thatthe retention of collagen is almost 100% during the same initial firstcritical hour. On the other hand, the dissolution from the Composite(-x-) in the initial hour is much higher and the collagen retentiondrops immediately to approximately 96% during that same critical firsthour although it stabilizes later on. FIG. 6 thus clearly demonstratesstability of the DSTI.

In order to determine the stability of the implant subjected totransport and handling, another study was performed with and withoutagitation and the dissolution of collagen from of DSTI under theseconditions was compared to the dissolution of collagen from thenon-lyophilized composite (Composite). Results are not shown.

These studies confirmed that even with agitation, there is a relativelysmall change in the accumulated release of protein into the solutionover a period of eight days but particularly during the first hourfollowing the rehydration.

2. Resistance to Change in Size and Shape

Another important feature of the DSTI is its resistance to change insize and shape. This feature is very important for implant efficacy asany change in the size and shape by shrinking or swelling can negativelyeffect the outcome of the implantation surgery. An implant that wouldget smaller by shrinking will not fill the defect, will not provide astructural support for migration of cells from the surrounding tissue orcell integration into the surrounding tissue and may also be dislodgedfrom the defect. Swelling of the implant could, on the other hand, causethe implant to swell within the defect, decrease the structural supportfor cells and be rejected or ejected from the defect because of itslarger size.

The resistance to change in shape and size means that for implantationinto a defect of discernable size, the functional construct must notswell or shrink extensively upon rehydration during time of preparationbefore surgery or after placement of the implant into the defect.

FIG. 7 presents the percent change in surface area of DSTI, rehydratedwith and maintained in culture for more than 24 hours in an aqueousphosphate buffered saline. The results seen in FIG. 7 show that there isvery little change in size and shape during the critical first hour inDSTI. In three lots of double-structured tissue implants subjected tothe dehydrothermal treatment there was approximately a 2-5% change insurface area within the first hour and such change was maintained withinthese parameters for more than 24 hours following the rehydration.

3. Cell Viability

Another important feature of the tissue implant is to provide supportand conditions for cell migration from surrounding tissue or for thecell integration into surrounding tissue in the case when the cells areseeded into the DSTI before implanting. This feature is determined bycell viability within the DSTI and provides another criteria fordetermining functionality and usefulness of the DSTI.

In order for an implant to be functionally viable, the implant mustprovide a structural support for cells as well as provide or permitconditions to be provided for cell seeding into the implant, cell growthwithin the implant and/or cell migration into or from the surroundingtissue.

Conditions for cell seeding, their growth within the implant, theirnutritional and metabolic needs are designed based on the type of cellsthat the implant is supposed to deliver and support. For example, if theimplant is designed for repair of a skin defect, the cells and theirrequirement will be different than if the implant is designed for repairof a chondral or bone lesion. Conditions for structural support andconditions for promotion of cell growth, their migration and/orintegration into the surrounding tissue will be adjusted based on thetissue where the DSTI will be implanted and the function the implantwill assume in repair of the tissue defect.

While the DSTI of the invention is preferably suitable for use intreatment and repair of chondral, subchondral or bone lesions, the DSTI,as such, is suitable to be used for repair of any other tissue or tissuedefect.

To determine the cell survival within the DSTI, studies were performedto determine the cell viability by determining their survival and growthwithin the DSTI. Cell viability was determined for three lots of DSTIthat had been seeded with chondrocytes after 1 day and 21 days inculture. Results are seen in FIG. 8.

FIG. 8 shows production of sulfated glycosaminoglycan and DNA(S-GAG/DNA) by chondrocytes seeded in double-structured tissue implant(DSTI) and in a composite (Composite) comprising a primary scaffoldloaded with a composition for a secondary scaffold before lyophilizationand dehydrothermal processing, after 14 days in culture.

Results seen in FIG. 8 demonstrate that inclusion of the secondaryscaffold within DSTI supports the growth of cells and deposition ofextracellular matrix measured here as production of sulfatedglycosaminoglycan. A comparison between the double-structured tissueimplant and the Composite showed comparable results with little evidenceof significant steric hindrance due to the added structural components.All samples had 96-100% viability at both timepoints indicating no celltoxicity. Furthermore, as with the DNA measurements, the total cellnumber increased over time which shows that the cells were retained inthe DSTI, and proliferated to fill the pores.

The successful implant, such as, for example, DSTI implanted into thecartilage lesion, must provide conditions allowing cells to form andgenerate the new extracellular matrix. In this regard, the implantporous structure must allow cells to migrate, be attached or aggregateinto and within the pores and function similarly to their normalfunction in the healthy tissue.

Consequently, the pore size of the implant and the consistency withrespect to pore size for the ingrowth of cells is important both forcell adhesion, extracellular matrix production and cell to cell contactand communication. Depending on the tissue to be repaired, the pore sizeof the primary and/or secondary scaffold will vary. For example,cartilage scaffolds would have an optimal pore size of approximately 200μm and bone would have a pore size in the range of 300 to 350 μm.

A significant advantage of having a double-structured tissue scaffoldarises from the increase in mechanical integrity relative to a primaryporous collagenous material because the polymerization createsfiber-like structure between the primary and secondary scaffold thatserves as a reinforcing network for cells.

In addition, due to inclusion of the secondary scaffold there is anincrease in overall surface area within the DSTI that permits cellsspreading and migration throughout the interstices of the DSTI. At thesame time, the secondary scaffold must be designed such that it is notof such high density that it becomes a blocking agent that acts as asteric hindrance for cell ingrowth and tissue repair.

As shown in FIGS. 4A, 4B and 5, double-structured tissue implant of theinvention provides optimal conditions for viability and growth of cells.

E. Process for Preparation and Use of the Double-Structured TissueImplant

The secondary scaffold is generated within confines of the primaryscaffold by a process comprising several stages and steps as set forthin Scheme 1. The process stages comprise pre-loading, loading,polymerization, treatment of composite double-structured scaffold,dehydrothermal treatment, packaging and, ultimately, its surgicaldelivery.

Scheme 1 Process for Production and Use of a Double-Structured ImplantStage 1—Pre-Loading

The pre-loading stage is a preparatory stage where the primary scaffoldis either obtained from commercial sources or is prepared according tothe procedure described in Example 1.

Step 1

Step 1 comprises obtaining or preparing a primary scaffold, typically acollagen containing honeycomb, sponge or lattice providing a structuralsupport for incorporation of the secondary scaffold.

In one embodiment, a bovine Type I collagen matrix with honeycomb (HC)like structure is obtained, for example, from Koken, Inc. (Japan) orfrom other commercial sources and used as primary scaffold. However,such commercially available honeycomb matrices have typically randomlydistributed pores of irregular shape and size. The pores of thesestructures are not always vertically positioned.

In another embodiment, and preferably, a primary honeycomb scaffold isproduced according to a process described in Example 1, wherein saidprimary scaffold has randomly or non-randomly oriented pores ofsubstantially the same size and shape.

The shape and size of the primary scaffold determines a size of thedouble-structured tissue implant (DSTI) ultimately delivered to thesurgeon for implantation into the tissue defect.

Typically the DSTI has a rectangular, circular or oval shape withdimensions of about 50 mm and a vertical thickness of about 1 to 5 mm,preferable 1-2 mm. Preferred dimensions of the DSTI and, therefore, thedimensions of the primary scaffold are 50×50 mm×1.5 mm, with poresoriented substantially vertically, said pores having a pore size of fromabout 100 to about 400 μm, preferably about 200±100 μm and pore lengthof 1.5 mm. However, dimensions of the primary scaffold may be any thatare required by the tissue defect to be repaired and that can beprepared by the process of the invention.

Step 2

Step 2 comprises preparing a composition for preparation of a secondaryscaffold (basic solution) and comprises neutralization of a solublecollagen solution having an initial acidic pH of about pH 1.5-4,preferably between about pH 1.9-2.2, a collagen concentration from about0.5 to about 10 mg/ml of collagen, preferably about 2.9 to about 3.2mg/ml, a surfactant concentration from about 0.05 to about 10 mg/ml,preferably about 0.29 to about 0.32 mg/ml and osmolality from about 20to about 400 mOsm/kg, preferably about 280 to about 320 mOsm/kg. Thesoluble collagen solution is then neutralized with any suitable baseand/or buffer to pH in a range from about pH 7.3 to about pH 7.7 toderive the basic solution. Preferably, the solution is neutralized byadjusting pH to neutrality 7.4 using a collagen/surfactant, 10 Dulbeccophosphate buffered saline (DPBS) and 0.1 M NaOH in 8:1:1 ratio or usingan aqueous solution or ammonia vapor in concentration sufficient toneutralize acid within the collagen solution. The final osmolality andpH of the basic solution is about 290 mOsm/kg and pH 7.4, respectively.

The suitable buffers for solubilization of the Type I collagen are, forexample, a formic acid containing buffer at pH 4.8, acetic acidcontaining buffer at pH 5.0 or a diluted hydrochloric acid containingbuffer at pH 3.0.

Neutralization is typically carried out using ammonia aqueous solutionor a vapor of about 0.3%-1% ammonia, or in concentration sufficient toneutralize the acidic pH over about 12 to about 24 hour period. Thisfactor has also been found to affect the collagen polymerization andformation of pores having homogeneous pore size.

Stage 2—Loading and Precipitation

The primary scaffold is loaded with a basic solution for the secondaryscaffold comprising soluble collagen solution containing a surfactant.This basic solution is subsequently precipitated within pores of theprimary scaffold.

Loading the primary scaffold with the basic solution for the secondaryscaffold is performed using any suitable method. Soaking, wicking,submerging the primary scaffold in the solution, electrophoresis and anyother suitable means. Once the basic solution for the secondary scaffoldis introduced into the primary scaffold, a composite of both issubjected to a process or treatment that results in formation of thesecondary scaffold inside pores of the primary scaffold.

Step 3

The neutralized basic solution of step 2 is loaded into the primaryscaffold by placing from about 3.75 to about 7.5 ml (approximately 1 to2×volume), preferably a volume about 4.9 ml (approx. 1.3×volume of theprimary scaffold) of the secondary scaffold basic solution on the bottomof a dish and then placing the primary scaffold in this solution andallowing it to be soaked up.

Stage 3—Polymerization of the Soluble Collagen within a Primary Scaffoldinto a Secondary Scaffold

The primary scaffold loaded with the neutralized basic solutioncomprising the soluble collagen and the surfactant is then subjected toconditions resulting in precipitation of the neutralized basic solutionwithin the pores of the primary scaffold thereby generating astructurally distinct secondary scaffold.

Typically, and allowing for variability of the basic solution orcomposition used for creating of the secondary scaffold, the compositionintroduced into the pores of the primary scaffold is gelled orprecipitated within said primary scaffold and may also be cross-linkedusing chemicals such as glutaraldehyde or another multifunctionalaldehyde, where the aldehyde reacts with amino groups of the collagenyielding a Schiff base, which can be stabilized by a reduction reaction;carbodiimide reagent, such as carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) with or without N-hydroxy-succinimide(NHS) where the HNS is used to suppress side reactions. Additionally,EDC and NHS can be used in combination with diamine or diacid compoundsto introduce extended cross-links; acyl azide where the acid areactivated and subsequently reacted with an adjanced amine group; epoxycompounds such as 1.4-butanediol diglycidyl ether, or cyanamide.

In addition, irradiation such as short wave length UV irradiation (254nm) can introduce cross-links in the collagen.

Step 4

The primary scaffold loaded with the neutralized collagen/surfactantsolution in a range from about 1 to about 2 volumes of the primaryscaffold is then placed in an incubator at a temperature from about 25°C. to about 38° C., preferably to about 37° C. temperature, typicallyfor from about 10 minutes to about 18 hours, more typically for about 20to about 100 minutes, preferably for about 40 to 60 minutes, and mostpreferably for a time when the precipitation of the neutralized collagensolution into a solid secondary scaffold occurs.

Step 5

In order to assure that the vast majority of the salt of theprecipitated collagen solution within the pores of the primary scaffoldis removed, a composite consisting of the primary scaffold having thesecondary scaffold precipitated within is subjected to a washing stepwhereby the majority of the salts are removed.

The composite (Composite) comprising the primary scaffold and thesecondary scaffold precipitated within, is washed by placing saidcomposite in a volume of from about 20 ml to about 10 liters, preferablyabout 500 ml, of de-ionized water further containing a non-ionicsurfactant. The surfactant is typically present in concentration fromabout 0.05 to about 1.0 mg/ml, preferably about 0.23 mg/ml. Mostpreferred surfactant is PLURONIC® F127.

Typically, the washing step takes approximately 30 minutes. There may beone or several washing step repetitions. All excess non-precipitatedcollagen is removed during the extraction from the composite into thewash solution.

Polymerizing of the collagen present in the secondary scaffold solutionloaded within the primary scaffold pores results in formation of a soliddouble-structured composite, as defined above, comprising the primaryscaffold and the secondary scaffold precipitated therewithin.

Following the precipitation or gelling and washing, the composite issubjected to lyophilization and dehydrothermal treatment.

Stage 4—Dehydration of Composite Double-Structured Scaffold

The solid double-structured composite is then dehydrated using anymethod suitable for such dehydration. Typically, such dehydration willbe freeze-drying or lyophilization. Freezing is typically carried out attemperature from about −10° C. to about −210° C., preferably from about−80° C., over a period of about 2 to about 60 minutes. Water removal(drying) is achieved by exposure to an anhydrous atmosphere from −20° C.to about 50° C. preferably at 37° C. for about 24 to about 48 hours. Thefreeze-dried composite is then lyophilized forming the LyophilizedComposite.

The gradual nature of the polymerization and slow process of waterremoval typically maintains the architectural elements of the secondaryscaffold and achieves the proper orientation and diameter of the pores.

Step 6

Dehydration is achieved with freezing the solid double-structuredcomposite by placing it on the metal shelf of a freezer and adjustingthe temperature to from about −10° C. to about −210° C., preferably toabout −60° C. to about −90° C., and most preferably for about −80° C.,for about 2 to about 60 minutes, preferably for about 20-45 minutes andmost preferably for about 30 minutes.

Step 7

The frozen and dehydrated solid double-structured composite is thensubjected to lyophilization. The frozen dehydrated composite is removedfrom the freezer and placed into a pre-cooled lyophilization chamber.Lyophilization typically occurs in about 15-21 hours, depending on thesize and shape of the composite but is typically and preferablycompleted in about 18 hours.

Stage 5—Dehydrothermal Treatment

To further stabilize the composite and to achieve necessary stability,resistance to dissolution and sterility of the final product, the soliddouble-structured composite is subjected to dehydrothermal (DHT)treatment. DHT treatment achieves cross-linking of the collagen with thesurfactant and at higher temperatures also sterilizes the DSTI.

Cross-linking step prevents dissolution of the secondary scaffold uponrehydration before or after implantation.

Step 8

This step is performed to sterilize and cross-link the double structuredtissue implant.

The lyophilized double-structured composite is placed into a dry glasschamber or container and covered with the glass, aluminum foil oranother suitable material resistant to higher temperatures. Thecontainer with the lyophilized double-structured composite is placedinto the pre-heated dehydrothermal oven and subjected to a temperaturein a range from about 70° C. to about 200° C., preferably from about130° C. to about 150° C., and most preferably about 135° C., undervacuum, for about 30 minutes to about 7 days, preferably for about 5-7hours and most preferably for about 6 hours.

Such treatment stabilizes the composite, makes it resistant to collagendissolution upon wetting, provides for rapid wetting and assures none orminimal shrinkage or swelling upon wetting with a physiological solutionor buffer, and sterilizes the double-structured tissue implant.

Stage 6—Packaging and Storage

The double-structured tissue implant fabricated by the process describedabove is then ready for a sterile packaging and storage. In this form,the DSTI has a long shelf-life.

Step 9

The double structured tissue implant is removed from the dehydrothermaloven and transferred aseptically into sterile environment, such as a BioSafety Cabinet (BSC), where it is packaged under conditions assuringsterility. The double-structured tissue implant is then ready to bestored at room temperature until its use.

Stage 7—Delivery by Implantation

Packaged double-structured tissue implant is delivered or made availableto a surgeon for implantation into a tissue defect.

Step 10

During surgery, surgeon determines an extent of the defect or lesion tobe repaired, opens the packaged product, cuts the DSTI to size of thedefects and places the cut-to-size piece into said defect. Typically,the implant is placed into the defect in a dry form and a suitablephysiologically acceptable solution is then added to wet the implant insitu. In alternative, the implant may be wetted before the implantationand then placed into the defect.

Since the implant is very stable, and does not change its size or shapesignificantly by shrinking or swelling, the implant fits tightly intothe defect or lesion. To assure that the implant stays within the defector lesion, such defect or lesion is first coated with a suitable tissueadhesive, sealant or glue that keeps the implant in place. Inalternative, the defect or lesion may be pretreated with microfracturewhere the tissue underlying the lesion or defect is microfractured withmicrochannels to permit the blood and nutrient supply into the lesion ordefect, lining the defect or lesion but not the microfracture, with theadhesive, glue or sealant and placing the implant as described above. Inboth instances, the implant placed into the lesion or defect mayoptionally be covered with another layer of the adhesive, sealant orglue.

In some instances, cells, drugs or modulators may be loaded into theDSTI or attached to the second scaffold before implantation and wetting,during wetting following the implantation, or independently providedafter the implantation.

Results obtained for three separate lots containing three rehydratedDSTIs per each lot, are seen in Table 2. The DSTI is rehydrated byplacing a droplet of phosphate buffer saline (1.5×volume of PBS), on topof the DSTI and the rehydration time is measured as the time it takesfor the DSTI to be completely hydrated.

TABLE 2 Number of Sample Results Attribute (n/lot) Lot #1 Lot #2 Lot #3Rehydration Time (seconds) 3 <2 <2 <2 Rehydrated pH 3  7.7 ± 0.1 7.8 ±0   7.7 ± 0.1 Rehydrated osmolality 3 317 ± 6  356 ± 4  319 ± 1 (mOsm/kg) Size variation at hydration (%) 3 99.8% ± 5.2% 100.6% ± 10.0%99.7% ± 2.3% Collagen Retention in PBS (%) 3 99.4% ± 0.2% 99.1% ± 0.1%99.2% ± 0.2%

As seen in Table 2, results obtained in three different lots in threedifferent studies are closely similar confirming the reproducibility ofthe process as well as consistency of the parameters observed afterrehydration.

The rehydration time for each lot is less then 2 second evidencing avery fast wettability of the DSTI products.

The pH of the rehydrated DSTI products is between 7.7 and 7.8 in alllots.

Osmolality of the rehydrated DSTI products is between 317 and 356mOsm/kg in all lots.

Variation in size of rehydrated DSTI products is negligible evidencingthat there is no shrinkage or swelling upon hydration of DSTI.

Collagen retention within the rehydrated DSTI is above 99%, evidencing agreat stability of the DSTI products.

III. Method of Use of Double-Structured Tissue Implant

Double-structured tissue implant of the invention is useful fortreatment and repair of tissue defects of various tissues. Suchtreatment is achieved by implanting the DSTI into the defect in surgicalsetting.

In this regard, the use of DSTI, as described herein in FIGS. 9A and 9Billustrates its implantation into the articular cartilage lesion.However, the same or similar process would be used for implantation ofthe DSTI into defect of any other tissue.

FIG. 9 is a schematic illustration of two treatment protocols forimplantation of the DSTI into a cartilage lesion injuries usingdouble-structured tissue implant. FIG. 9A shows the treatment with DSTIwithout microfracture, FIG. 9B shows treatment with microfracturepretreatment. FIGS. 9A and 9B demonstrate how the double secondaryscaffold will be applied in a surgical operating room setting. Theinitially oversized double-structured tissue implant (DSTI) is first cutand trimmed to match the defect. A physiologically acceptable tissueadhesive is applied to the defect to coat the defect (FIG. 9A) or thesubchondral plate of the defect is penetrated by microfracture, adhesiveis applied between microfracture penetrations (FIG. 9B) and the precutsized DSTI is placed into the defect. The DSTI is then rehydrated with aphysiologically acceptable solution optionally containing agents thatstimulate healing. The DSTI is then sealed within the defect usinganother layer of the tissue adhesive to form a final bonding.

A. Adhesives and Tissue Sealants

The double-structured tissue implant is implanted into a tissue defector cartilage lesion covered with a biocompatible adhesive, tissuesealant or glue. Typically, the sealant is deposited at and covers thebottom of the defect or lesion and may also be used to cover the implantafter implantation.

Generally, the tissue sealant or adhesive useful for the purposes ofthis application has adhesive, or peel strengths at least 10 N/m andpreferably 100 N/cm; has tensile strength in the range of 0.2 MPa to 3MPa, but preferably 0.8 to 1.0 MPa. In so-called “lap shear” bondingtests, values of 0.5 up to 4-6 N/cm² are characteristic of strongbiological adhesives.

Such properties can be achieved by a variety of materials, both naturaland synthetic. Examples of suitable sealant include gelatin anddi-aldehyde starch described in PCT WO 97/29715, 4-armed pentaerythritoltetra-thiol and polyethylene glycol diacrylate described in PCT WO00/44808, photo-polymerizable polyethylene glycol-co-poly(a-hydroxyacid) diacrylate macromers described in U.S. Pat. No. 5,410,016,periodate-oxidized gelatin described in U.S. Pat. No. 5,618,551, serumalbumin and di-functional polyethylene glycol derivatized withmaleimidyl, succinimidyl, phthalimidyl and related active groupsdescribed in PCT WO 96/03159.

Sealants and adhesives suitable for purposes of this invention includesealants prepared from gelatin and dialdehyde starch triggered by mixingaqueous solutions of gelatin and dialdehyde starch which spontaneouslyreact and/or those made from a copolymer of polyethylene glycol andpolylactide, polyglycolide, polyhydroxybutyrates or polymers of aromaticorganic amino acids and sometimes further containing acrylate sidechains, gelled by light, in the presence of some activating molecules.

Another type of the suitable sealant is 4-armed polyethylene glycolderivatized with succinimidyl ester and thiol plus methylated collagenin two-part polymer compositions that rapidly form a matrix where atleast one of the compounds is polymeric, such as polyamino acid,polysaccharide, polyalkylene oxide or polyethylene glycol and two partsare linked through a covalent bond, for example a cross-linked PEG withmethylated collagen, such as a cross-linked polyethylene glycol hydrogelwith methylated collagen.

Preferable sealants are 4-armed tetra-succinimidyl ester PEG,tetra-thiol derivatized PEG and PEG derivatized with methylated collagen(known as CT3), commercially available from Cohesion Inc., Palo Alto,Calif. and described in U.S. Pat. Nos. 6,312,725B1 and 6,624,245B2 andin J. Biomed. Mater. Res., 58:545-555 (2001), J. Biomed. Mater. Res.,58:308-312 (2001) and The American Surgeon, 68:553-562 (2002), allhereby incorporated by reference.

Sealants and adhesives described in copending U.S. application Ser. No.10/921,389 filed Aug. 18, 2004 and Ser. No. 11/525,782 filed Dec. 22,2006, are hereby incorporated by reference.

B. Use of DSTI for Treatment of Chondral Defects

One example of utility of the DSTI is its use for treatment of chondraldefects.

To be successful for treatment of articular cartilage, the DSTI mustprovide conditions allowing the chondrocytes or mesenchymal stem cellsseeded therein to be able to form and generate the new extracellularmatrix. In this regard, the DSTI pore structure must allow cells tomigrate into the pores and function similarly to their normal functionin the healthy tissue. The extracellular matrix formed by the cellsseeded within the DSTI then provides means for growing a new hyaline orhyaline-like cartilage for treatment, replacement or regeneration of thedamaged or injured articular cartilage. Such treatment is currentlydifficult because of the unique properties of the articular cartilagethat is not the same as and does not behave as other soft tissues.

C. Use of DSTI for Treatment of Other Conditions

In addition to cartilage repair, a number of other chronic conditionsrepresent instances where the implantation of the double structuredscaffold can provide a clinically important bridge for tissue repair.

For example, genitourinary tissues have been fabricated from a varietyof materials. The DSTI once placed at the site of tissue damage willprovide a support for development of new tissues occurs in accordancewith predefined configuration. In these applications, similar tocartilage, the DSTI must resist the dynamic forces generated by thesurrounding muscle and connective tissues and maintaining its structureduring a necessary period of cellular infiltration and tissue formation.

The rapidity by which tissue differentiation and structural integrityare established is subject to modulation through the use specificsignaling factors localized within the primary and secondary collagenouscomposite. Although the limits by which, for example, new muscleformation can be derived from progenitor cells, suggests thatlocalization of the mesenchymal cells to the site of damage in responseto homing molecules, such as chemokines and cell receptor ligands, maybe used to accelerate repair of muscle, either cardiac or skeletal. DSTImay be used to deliver these cells or modulators to the cite of damage.

Finally, wound healing applications have remained a primary goal in theuse of tissue implants for cell-based tissue repair. Treatment of acuteand chronic wounds is dependent on a multi-faceted transition by whichprogenitor cells encounter soluble mediators, formed blood elements,extracellular matrix macromolecules and parenchymal cells that thenserve to reestablish a body surface barrier through epithelialization.In this instance either the double-structured scaffold or the standalone secondary scaffold implant may provide a novice stromal layer intowhich blood vessels and progenitor cells can migrate. From thismigration, the progenitor cells may then undergo differentiation intothe fibroblast stromal cell and generate or recruit epithelial cells tosupport reestablishment of dermal and epidermal layers at the time ofwound closure.

D. Basic Requirements for DSTI

The collagen-based primary and secondary scaffolds of the DSTI areessential components of the DSTI and are responsible for capability ofDSTI to initiate the repair and induction of repair of tissue defects.

The first requirement is that the scaffolds are prepared from thebiocompatible and preferably biodegradable materials that are the sameor similar to those observed in the tissues to be repaired, hence theinstant DSTI are prepared from collagen or collagen-like materials.

The second requirement is that the scaffolds have a spatial organizationand orientation similar to that of the tissue to be repaired. The porousstructure of both primary and secondary scaffold provides suchorganization.

The third requirement is that the scaffold have a pore densitypermitting the seeding of the cells into said scaffolds in numbersneeded for initiation of a tissue recovery or formation of new tissue invivo. The substantially homogeneous pore size and distribution withinthe DSTIs allows the cell seeding and assures cell viability.

The fourth requirement is that the scaffolds have sufficient number ofpores for the number of cells needed for initiation of the tissuerecovery and repair. The spatial organization of both scaffolds haveoptimized number of pores.

The fifth requirement is that the pores have substantially the same sizeand that such size is substantially the same from the top apical to thebottom basal surface of the pores, said pores being organizedsubstantially vertically from the top to the bottom. The primaryscaffold have such organization.

The sixth requirement is stability of the DSTI. The double-structuredorganization of the DSTI provides such stability during wetting,reconstitution, resistance to dissolution and to shrinkage or swelling.

The seventh requirement is that DSTI provides support and conditions forcell migration from the surrounding tissue, for integration of seededcells into the surrounding tissue and generally that assures the cellviability. The DSTI provides such conditions and the cells seeded withinDSTI have almost 100% viability.

EXAMPLE 1 Preparation of the Primary Scaffold

This example describes one exemplary method for preparation of thecollagen-based primary scaffold suitable as a structural support forpreparation of the DSTI.

Type I collagen is dissolved in a weak hydrochloric acid solution at pH3.0 with the collagen concentration and osmolality of the solutionadjusted to about 3.5 mg/ml and 20 mOsm/kg, respectively. The solution(70 ml) is poured into a 100 ml Petri dish and the Petri dish containingthe collagen solution is centrifuged at 400×g for 30 minutes to removeair bubbles. Neutralization and subsequent precipitation or gelling iscarried out in a 7 liter chamber containing 10 ml of 15% ammoniasolution over a 45 minutes period. The precipitated collagen is thenwashed in a large excess of de-ionized water. The water is changed asmany times as needed over next 3 days to remove formed salts and excessammonia.

The solution is then subjected to unidirectional freezing over a periodof about 4 hours. The Petri dish is placed on a stainless steel discwhich is partially submerged in a cooling bath. The temperature of thecooling bath is increased from 0° C. to −18° C. at a rate of 0.1°C./minute. The frozen water is removed by lyophilization over a periodof about 3 days. The lyophilized primary scaffold is thendehydrothermally (DHT) treated at 135° C. for about 18 hours beforebeing precut into slices of an appropriate thickness.

The organization of the newly synthesized cartilage specific matrixwithin the porous type I collagen is visualized and quantified usinghistological and image analysis methods.

EXAMPLE 2 Preparation of a Basic Solution for a Secondary Scaffold

This example describes preparation of the basic solution used forformation of the secondary scaffold. The basic solution comprises asoluble collagen in admixture with a PLURONIC® surfactant. The basicsolution is incorporated into the primary scaffold and processes intothe double scaffold tissue implant or processed as a stand aloneimplant.

Solution for the secondary scaffold is prepared by mixing PLURONIC® F127(2.32 mg, 0.29 mg/ml), obtained commercially from BASF, Germany, with 8ml of a solution containing 2.9 mg/ml bovine type I collagen dissolvedin hydrochloric acid (pH 2.0) at room temperature. The resultingsolution is neutralized with 1 ml of 10× Dulbecco's phosphate bufferedsaline (DPBS) and 1 ml of 0.1M NaOH to the final pH of 7.4.

In the alternative, the neutralization is achieved by ammonia aqueoussolution or ammonia vapor in concentration sufficient to neutralize acidwithin the collagen solution.

EXAMPLE 3 Preparation of the Secondary Scaffold as Stand Alone Unit

This example illustrates preparation of the secondary scaffold as astand alone implant or stand alone unit. For preparation of the standalone secondary scaffold, the basic solution prepared in Example 3 issubjected to precipitation or gelling followed by dehydrothermaltreatment.

The basic solution (2 ml) comprising collagen and PLURONIC® surfactantis placed in a small glass beaker and the beaker is placed into achamber (approximately 9 liters) charged with 1% ammonia solution. Thebasic solution is allowed to precipitate in the chamber over a period of15 minutes. The gelled or precipitated collagen is then washed in 500 mlof de-ionized water over a period of 30 minutes. The washing step isrepeated three times. The washed gel or precipitate is placed on metalshelf of a freezer at −80° C. over a period of 30 minutes. The frozengel or precipitate is removed from the freezer and lyophilized.Lyophilization is performed over a period of 10 hours. The lyophilizedconstruct is then dehydrothermally treated at 135° C. under vacuum for aperiod of 6 hours to form the secondary scaffold alone.

EXAMPLE 4 Preparation of the Double-Structured Tissue Implant

This example describes preparation of the double-structured tissueimplant (DSTI). The preparation of DSTI includes incorporation of abasic solution for formation of a secondary scaffold within the primaryscaffold and its further processing into DSTI.

4.9 ml (1.3×volume of the primary scaffold) of the neutralized basiccollagen/PLURONIC® solution prepared in Example 2, is placed in a dishand a primary scaffold, prepared in Example 1, precut into a squarehaving 50×50×1.5 mm dimensions is then placed into the neutralized basicsolution for the secondary scaffold. The basic neutralized solution isabsorbed into the primary scaffold by wicking or soaking.

The primary scaffold containing the neutralized solution is then placedin a 37° C. incubator over a period of 50 minutes to precipitate or gelthe neutralized collagen solution. The composite consisting of theprimary scaffold with the gelled or precipitated neutralized solutionwithin is then washed in 500 ml of de-ionized water over a period of 30minutes. The washed composite is placed on metal shelf of a freezer at atemperature −80° C. over a period of 30 minutes. The frozen composite isremoved from the freezer and lyophilized.

Lyophilization is performed over a period of 18 hours. The lyophilizedcomposite is then dehydrothermally treated at 135° C. under vacuum for aperiod of 6 hours to form the double-structured tissue implant (DSTI).

The DSTI is removed from the dehydrothermal oven and transferredaseptically into a Bio Safety Cabinet (BSC) where it is packaged.

EXAMPLE 5 Determination of Retention of Collagen within DSTI

This example describes a procedure used for determination of thestability of the double-structured tissue implant in vitro.

Three lots of DSTIs are prepared as described in Example 4 and cut to asize of 1.5×1.5×0.15 cm. Cut DSTIs are placed in 35 mm Petri dishes,rehydrated with 450 μl of phosphate buffered saline and additional 2 mlof phosphate buffered saline are added to each Petri dish containing theDSTI. The analysis for each lot consist of three replicates for a totalof 9 samples for the three lots.

Dishes containing individual DSTIs are placed in the incubator for theduration of testing. In the predetermined intervals of zero hour, 1hour, 3 days, 7 days and 14 days, 1 ml aliquot of the phosphate bufferedsaline is removed from each plate. Each removed 1 ml is replaced with 1ml of a fresh phosphate buffer saline. The removed aliquots aresubjected to a calorimetric protein assay for quantification of totalcollagen released into the saline.

Cumulative collagen retention curves are generated by subtraction of theamount of collagen released into the solution from the theoreticalcollagen load estimated at 0.777 mg of collagen/DSTI sample. Results areseen in FIG. 6.

EXAMPLE 6 Determination of Change of Size and Shape of DSTI FollowingRehydration

This example describes the retention of size and shape ofdouble-structured tissue implant in a phosphate buffered saline solutionover time.

The DSTI samples obtained in Example 5 are photographed at thedesignated intervals and the images generated are measured by ImageJ,publicly available Java-based image processing program developed at theNIH. The photograph of each sample at each time point is imported intoImageJ and the region of interest (DSTI area) is manually defined. Thearea was measured and the percent change is determined by dividing theareas at each time point by the rehydrated DSTI at time 0 andmultiplying by 100.

Results are seen in FIG. 7.

EXAMPLE 7 Studies of Biocompatibility

This example describes procedures used to determine cellbiocompatibility with the primary scaffold and with the DSTI.

The primary scaffold and DSTIs are prepared in three lots as describedin Examples 1 and 4. The chondrocytes are loaded into the three samplesof primary scaffolds and into the three lots of three samples each ofDSTIs. Time intervals for biochemical, image and cell viabilitydetermination is set to Day 0 (24 hours) and Day 21 (21 days) ofincubation in the culture medium.

The time and group protocol is seen in Table 3.

TABLE 3 Chondrocyte Compatibility Experimental Groups Sample NumbersGroup Description Time point Biochemistry Images Viability A DSTI #1 Day0 3 2 3 B DSTI #1 Day 21 3 2 3 C DSTI #2 Day 0 3 2 3 D DSTI #2 Day 21 32 3 E DSTI #3 Day 0 3 2 3 F DSTI #3 Day 21 3 2 3 G Primary Day 0 3 2 3Scaffold H Primary Day 21 3 2 3 Scaffold

DSTI—5 mm disks are cut from each lot of sheets. Primary scaffold 5 mmdisks are cut from primary scaffold.

The disks of the primary scaffold are loaded with the chondrocytesdissolved in a collagen solution at a cell concentration of 5×10⁶cells/ml. The primary scaffold does not contain the surfactant and isnot subjected to lyophilization or to a dehydrothermal treatment.

Both the primary scaffold and DSTI disks (total 24) are placed in 24well plates. Chondrocytes are seeded into the disks by the addition of20 μl of 3D cell culture medium (DMEM/F-12+10% FBS+1% ITS) at a cellconcentration of 5×10⁶ cells/ml. The disks loaded with cells are placedin the incubator for 1 hour at 37° C. and in 5% CO₂. Then 400 μl ofmedium is added and the plates are placed in the low oxygen incubatorovernight. At 24 hours (Day 0), one set of samples from each lot isremoved from culture. The remaining disks loaded with chondrocytes aretransferred to 12 well plates with 2 ml of 3D culture medium and aremaintained in culture at 37° C., 5% CO₂, 2% O₂ for three weeks withmedium changes once a week.

The primary scaffold disks are processed in the same way as DSTI disks.

Evaluations include assessment of chondrocyte growth, viability andphenotypic S-GAG expression in both the primary scaffold and DSTIs.Results are seen in FIG. 4B, and FIG. 5 for DSTI images, FIG. 8 forS-GAG and DNA production and in Table 1 for cell viability.

EXAMPLE 8 Production of S-GAG/DNA

This example describes conditions used for evaluation of production ofS-GAG and DNA by seeded chondrocytes.

DSTI and primary scaffold disks are prepared as described in Examples 1and 4 and seeded with 200,000 chondrocytes in 20 μl of 3D culture mediumby absorption at 37° C. for 1 hour. 400 μl of medium is added andincubated overnight. Disks are removed for analysis at predeterminedintervals.

At termination, the disks are placed in papain digest solution andincubated at 60° C. overnight. An aliquot of the digest from each diskis taken to measure S-GAG by the dimethylmethylene blue assay. Analiquot from each disk is taken for measurement of DNA by the Hoechstdye method. Results are shown in FIG. 8.

EXAMPLE 9 Determination of Viability of Chondrocytes

This example describes procedure used for determination of viability ofchondrocytes seeded in the DSTIs or in the primary or secondaryscaffolds.

DSTI, primary scaffold or secondary scaffold disks are seeded withapproximately 200,000 chondrocytes dissolved in the 20 μl of 3D culturemedium by absorption and incubated at 37° C. for 31 hours. 400 μl ofmedium is added and incubation is continued overnight.

Cell loaded disks are removed for analysis and 2 ml of medium is addedto remaining disks for continued incubation for 21 days.

At day 21, the chondrocyte-loaded DSTIs and the primary and secondaryscaffolds disks are placed in 1.5 ml microcentrifuge tubes and furtherincubated overnight in 0.15% collagenase. The digest is centrifuged at2000 rpm for 5 minutes and the supernatant aspirated. An aliquot ofculture medium (0.1 ml) is added to the cell pellets and an aliquottaken for counting. Cell viability and total cell count is determinedusing trypan blue. Results are shown in Table 1.

1. A double-structured tissue implant (DSTI) comprising a primaryscaffold and a secondary scaffold integrated into said first scaffoldwherein said primary scaffold is a porous structure prepared from abiocompatible material, said porous structure comprising randomly andnon-randomly organized pores and providing a structural support for thesecondary scaffold incorporated therein.
 2. The DSTI of claim 1 whereinsaid secondary scaffold is integrated into said primary scaffold byintroducing a composition comprising a soluble collagen,collagen-containing or collagen-like compound in combination with anon-ionic surfactant, into said pores of said primary scaffold,stabilizing said composition within pores of said primary scaffold byprecipitation or gelling and subjecting a resulting composite tolyophilization and dehydrothermal treatment.
 3. The DSTI of claim 2wherein said biocompatible material suitable for preparation of theprimary scaffold is selected from the group consisting of Type Icollagen, Type II collagen, Type IV collagen, gelatin, collagencontaining agarose, collagen containing hyaluronan, collagen containingproteoglycan, glycosaminoglycan, glucosamine, galactosamines,glycoprotein, collagen containing fibronectin, collagen containinglaminin, collagen containing bioactive peptide growth factors, collagencontaining cytokine, collagen containing elastin, collagen containingfibrin, collagen containing polylactic, polyglycolic or polyamino acid,collagen containing polycaprolactone, polypeptide gel, a copolymersthereof, a precursor thereof and combination thereof, wherein saidprecursor is selected from the group consisting of alpha 1 (Type I)peptide, alpha 2 (Type I) peptide, 2 (alpha 1, Type I) peptide, 1 (alpha2, Type I) peptide, 3 (alpha 1, Type II) and a combination thereof. 4.The DSTI of claim 3 wherein said soluble collagen, collagen-containingor collagen-like compound used for preparation of the composition forthe second scaffold is Type I collagen, Type II collagen, methylatedcollagen, gelatin or methylated gelatin.
 5. The DSTI of claim 4 whereinsaid non-ionic surfactant used for preparation of the composition forthe second scaffold is a PLURONIC®-type or a TRITON®-type surfactantcomprising polyethylene oxide with terminal oxide groups.
 6. The DSTI ofclaim 5 wherein said surfactant is a derivatized polyethylene glycol ora block co-polymer of polyoxyethylene (PEO) and polyoxypropylene (PPO)having the generic organization of polymeric blocks PEG-PPO-PEG orPPO-PEG-PPO.
 7. The DSTI of claim 6 wherein said surfactant is TRITON®X100, namely polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylether, or PLURONIC® F127, namely a polymer of polyoxyethylene (PEO) andpolyoxypropylene (PPO) with two 96-unit hydrophilic PEO chainssurrounding one 69-unit hydrophobic PPO chain.
 8. The DSTI of claim 2wherein said integration of the secondary scaffold into the primaryscaffold results in the double-structured tissue implant comprising twostructurally and functionally distinct sections, wherein one or bothsections may independently be seeded with cells or incorporated with apharmaceutical agent or modulator.
 9. The DSTI of claim 8 suitable forimplantation as an acellular implant or an implant comprisingchondrocytes for treatment and repair of articular cartilage injuries ordamage wherein said implant, following reconstitution and rehydrationwith a physiologically acceptable solution or buffer during surgery, isresistant to dissolution, to change in size and shape, maintains itscollagen retention, pH and osmolality and has a rehydration andwettability time within several seconds.
 10. A method for use of thedouble-structured tissue implant (DSTI) for implantation for treatmentof tissue defects and injuries, said method comprising steps: a.obtaining or preparing the double-structured tissue implant; b.preparing a tissue defect for implantation of said DSTI; c. precuttingthe DSTI into a size of the defect; d. coating the defect with a tissueadhesive; e. implanting the DSTI into said defect; f. rehydrating theDSTI with a physiologically acceptable solution or buffer; and g.covering said implant with a tissue adhesive.
 11. The method of claim 10wherein said double-structured tissue implant (DSTI) comprises a primaryscaffold and a secondary scaffold integrated into said first scaffoldwherein said primary scaffold is a porous structure prepared from abiocompatible material, said porous structure comprising vertically andnon-randomly organized pores and providing a structural support for thesecondary scaffold incorporated therein.
 12. The method of claim 11wherein in said DSTI, said secondary scaffold is integrated into saidprimary scaffold by introducing a composition comprising a solublecollagen, collagen-containing or collagen-like compound in combinationwith a non-ionic surfactant into said pores of said primary scaffold,stabilizing said composition within pores of said primary scaffold byprecipitation or gelling and subjecting a resulting composite tolyophilization and dehydrothermal treatment.
 13. The method of claim 12wherein said cells are chondrocytes, fibroblasts or mesenchymal stemcells.
 14. The method of claim 13 used for treatment of articularcartilage, meniscus, tendon or ligament defects.
 15. The method of claim14 wherein said defect is the cartilage defect further pre-treated withmicrofracture wherein said adhesive is placed into spaces between themicrofracture penetration and wherein said adhesive is a PEG derivatizedwith methylated collagen.
 16. A stand alone secondary scaffold tissueimplant comprising a basic solution comprising a soluble collagen,collagen-containing or collagen-like compound in combination with anon-ionic surfactant, wherein said basic solution is neutralized to pHabove 7.4%, precipitated, lyophilized and dehydrothermally treated. 17.A process for preparation of the double-structured tissue implant, saidprocess comprising steps: a. preparing a primary scaffold from abiocompatible material selected from the group consisting of Type Icollagen, Type II collagen, Type IV collagen, gelatin, agarose, collagencontaining hyaluronan, collagen containing proteoglycan,glycosaminoglycan, glucosamine, galactosamines or glycoprotein, collagencontaining fibronectin, collagen containing laminin, collagen containinga bioactive peptide growth factor, collagen containing cytokine,collagen containing elastin, collagen containing fibrin, collagencontaining polylactic, polyglycolic or polyamino acid, polypeptide gel,a copolymers thereof, a precursor thereof and combination thereof,wherein said precursor is selected from the group consisting of alpha 1(Type I) peptide, alpha 2 (Type I) peptide, 2 (alpha 1, Type I) peptide,1 (alpha 2, Type I) peptide, 3 (alpha 1, Type II) and a combinationthereof, as a porous structure having vertical orientation of the pores;b. preparing a composition for secondary scaffold wherein saidcomposition comprises an acidic solution comprising a soluble collagen,collagen-containing or collagen-like compound in combination with anon-ionic surfactant; c. introducing said composition into pores of saidprimary scaffold; d. stabilizing said composition within said pores ofsaid primary scaffold by neutralization, precipitation or gellingthereby producing a composite of the primary and the secondaryscaffolds; e. removing non-precipitated collagen and dehydrating saidcomposite by freeze-drying; f. lyophilizing said composite into alyophilized composite; and g. subjecting said lyophilized composite to adehydrothermal treatment.
 18. The process of claim 17 wherein saiddehydrothermal treatment comprises subjecting said lyophilized compositeto temperatures in a range from about 70° C. to about 200° C. undervacuum, for about 30 minutes to about 7 days.
 19. The process of claim18 wherein said dehydrothermal treatment comprises subjecting saidlyophilized composite to temperatures from about 130° C. to about 150°C. for about 5-7 hours.
 20. The process of claim 19 wherein saidneutralization step comprises neutralization of the composition forproduction of the secondary scaffold to pH in a range from about pH 7.3to about pH 7.7; said precipitation step of the neutralized compositionwithin the pores of the primary scaffold is by incubating said compositeat a temperature from about 4° C. to about 38° C., for a time needed forprecipitation of the neutralized composition into a solid secondaryscaffold within said composite; said removal of non-precipitatedcollagen is by washing said composite; said dehydration step is byfreeze-drying carried out at temperature from about −40° C. to about−90° C. over a period of about 2 to about 12 hours; and saidlyophilization step is achieved by introducing said composite into apre-cooled lyophilization chamber for about 15-21 hours.